Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) infection is an important risk factor for development of shipping fever pneumonia in feedlot cattle, and infects but does not cause morphologic evidence of damage to airway epithelial cells. We hypothesized that BVDV predisposes to bacterial pneumonia by impairing innate immune responses in airway epithelial cells. Primary cultures of bovine tracheal epithelial cells were infected with BVDV for 48 h, then stimulated with LPS for 16 h. Expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) mRNA was measured by quantitative RT-PCR, and lactoferrin concentrations were measured in culture supernatant by ELISA. BVDV infection had no detectable effect on the constitutive expression of TAP and LAP mRNA or lactoferrin concentration in culture supernatant. LPS treatment provoked a significant increase in TAP mRNA expression and lactoferrin concentration in the culture supernatant (p<0.01), and these effects were significantly (p<0.02, p<0.01) abrogated by prior infection of the tracheal epithelial cells with the type 2 ncp-BVDV isolate. In contrast, infection with the type 1 ncp-BVDV isolate had no effect on TAP mRNA expression or lactoferrin secretion. LPS treatment induced a significant (p<0.001) upregulation of LAP mRNA expression, which was not significantly affected by prior infection with BVDV. These data indicate that infection with a type 2 BVDV isolate inhibits the LPS-induced upregulation of TAP mRNA expression and lactoferrin secretion by tracheal epithelial cells, suggesting a novel mechanism by which this virus abrogates respiratory innate immune responses and predisposes to bacterial pneumonia in cattle.

Efficacy of tulathromycin compared with tilmicosin and florfenicol for the control of respiratory disease in cattle at high risk of developing bovine respiratory disease.

Three studies conducted at feedlots in Colorado, Idaho, and Texas examined the comparative efficacy of tulathromycin injectable solution for the treatment of cattle at high risk of developing undifferentiated bovine respiratory disease (BRD). Each study randomly allocated 250 calves to receive tulathromycin at 2.5 mg/kg and 250 calves to receive either tilmicosin at 10 mg/kg (Colorado site) or florfenicol at 40 mg/kg (Idaho and Texas sites) on arrival at the feedlot. Calves were housed by treatment group in pens with 50 calves/pen. Beginning 3 days after antimicrobial treatment, cattle were observed for signs of BRD daily until harvest. In all three studies, the treatment success rates at 28 days after treatment and at harvest were significantly higher (P < or = .013) for cattle treated with tulathromycin than for cattle treated with either tilmicosin or florfenicol. Fewer tulathromycin-treated cattle were removed from the group as "chronics" or "mortalities" at 28 days posttreatment (P < or = .014) in all three studies. Tulathromycin demonstrated superior efficacy compared with tilmicosin and florfenicol when treating groups of high-risk cattle before the onset of signs of BRD.

Clinical effectiveness of tulathromycin, a novel triamilide antimicrobial, for the control of respiratory disease in cattle at high risk for developing bovine respiratory disease.

The efficacy of tulathromycin in decreasing the incidence of morbidity and mortality due to bovine respiratory disease (BRD) in 1,239 high-risk cattle was investigated at four US feedlots. Calves not exhibiting clinical signs of BRD received one of three treatments administered subcutaneously in the neck: physiologic saline at 0.02 ml/kg, tulathromycin at 2.5 mg/kg, or tilmicosin at 10 mg/kg. Each treatment group consisted of 413 calves. Calves with clinical signs of BRD and rectal temperatures of 104 degrees F or higher on days 1 through 14 were considered treatment failures (BRD morbidity). Nasopharyngeal swabs from saline-treated morbidities were submitted for isolation and identification of BRD organisms. Respiratory disease morbidity was highest in calves treated with saline and significantly (P < or = .0001) lower in calves administered tulathromycin or tilmicosin. Morbidity from BRD was significantly (P < or = .0001) higher in calves treated with tilmicosin than in calves treated with tulathromycin. Under conditions of this study, tulathromycin, given to calves at high risk of developing BRD, was significantly more effective in reducing BRD morbidity when compared to both saline- and tilmicosin-treated calves.

Ultrastructural study of ovine pulmonary pasteurellosis: involvement of neutrophils and macrophages.

Pasteurellosis is a common infectious disease characterised by fibrinous pneumonia and involving neutrophils and macrophages. This study aimed to determine the timing and extent of the pathogenic involvement of these cell elements in lesions induced in experimentally-infected lambs. A concentration of approximately 3x10(8) bacteria/ml. was inoculated into 30 two-month-old disease-free Merino lambs. Five lambs were assigned to each of five experimental batches, slaughtered on days 1, 3, 7, 11 and 15 following intratracheal inoculation, and to one control batch inoculated with a sterile solution. One control animal was slaughtered at the same time as each experimental batch. More characteristic lesions occur in bronchioles, peribronchial tissue and alveoli and are characterised by fibrinous processes. From the start of the experiment, epithelial-cell disruption and loss of microvilli were apparent; cell debris, desquamate cells and bacterial elements were observed in bronchiolar lumina, embedded in a fibrillar granular material. Alveolar structures displayed fewer neutrophils and macrophages, containing phagocytic vacuoles. Laminar bodies were apparent in type II pneumocytes. The interseptal area contained similar cell types, as well as abundant edema. In the course of the experiment, macrophage numbers increased in all the areas involved, with signs of intense phagocytic activity. The final phase of the experiment was characterised by a mild interseptal infiltrate and by clear alveolar lumina.

Increased tumor necrosis factor-alpha and interleukin-1 beta expression in the lungs of calves with experimental pneumonic pasteurellosis.

We used a well characterized pneumonic pasteurellosis model in calves to determine whether increased proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) expression and secretion were associated with pneumonic lesions. Bronchoalveolar lavage fluids, lavage cells consisting of alveolar macrophages and neutrophils with degenerative changes, and lung tissues were analyzed for the presence of TNF-alpha and IL-1 beta approximately 48 h following endobronchial inoculation of logarithmic phase Pasteurella haemolytica 12296 organisms. Levels of TNF-alpha and IL-1 beta mRNA were significantly increased in lavage cells of P. haemolytica-infected animals but not in cells from phosphate buffered saline (PBS) inoculated controls based on in situ hybridization analysis. Significantly increased levels of TNF-alpha, and IL-1 beta mRNA were also expressed within the pneumonic lesions from P. haemolytica-infected calves. In contrast, lung tissues from PBS-inoculated control calves had cytokine mRNAs expressed at extremely low levels. Increased levels of bioactive IL-1 and immunoreactive (not bioactive) TNF-alpha were found in lavage fluids from P. haemolytica-infected calves compared with lavage fluids from PBS-inoculated calves. These findings indicate that the proinflammatory cytokines TNF-alpha and IL-1, may be associated with pathogenesis of lung injury in bovine pneumonic pasteurellosis.

Role of Mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis.

Bovine pneumonic pasteurellosis continues to be a major respiratory disease in feedlot cattle despite the recent advances in our understanding of the underlying complexities of causation. The etiological agent, Mannheimia haemolytica, possesses several virulence factors, including capsule, outer membrane proteins, adhesins, neuraminidase, endotoxin and exotoxic leukotoxin. Accumulating scientific evidence implicates leukotoxin as the primary factor contributing to clinical presentation and lung injury associated with this disease. Unlike other virulence factors, leukotoxin shows cell-type- and species-specific effects on bovine leukocytes. Recent investigations have delineated the mechanisms underlying the target-cell-specificity of leukotoxin and how this contributes to the pathogenesis of lung damage. This review summarizes current understanding of the secretion, regulation, mechanisms of action and evolutionary diversity of leukotoxin of M. haemolytica. Understanding the precise molecular mechanisms of leukotoxin is critical for the development of more effective prophylactic and therapeutic strategies to control this complex disease.

Experimental production of bovine pneumonic pasteurellosis.

Pneumonic pasteurellosis has been reproduced in conventional, weaned, Friesian-cross calves using a strain of Pasteurella haemolytica biotype A, serotype 1 (P haemolytica A1) isolated from a pathologically confirmed incident of bovine pneumonic pasteurellosis. The major clinical findings were pyrexia, hyperpnoea, tachypnoea, nasal discharge and reduced appetite. Fibrinous pneumonia was present in the lungs of animals at necropsy on days 2 and 3 after initial infection while by days 9 and 10 after initial infection many of the areas of fibrinous pneumonia were confined by a fibrous capsule forming well defined nodules. During the experiment natural transmission of the infecting strain of P haemolytica A1 occurred in two control calves which developed a condition identical to that in the artificially infected calves. P haemolytica A1 was repeatedly recovered from the nasopharynx of infected calves and at necropsy throughout the upper and lower respiratory tracts. Seroconversion, as measured by indirect haemagglutination, to the organism developed in all infected calves by days 9 and 10 after initial infection. The clinical, microbiological and pathological findings were identical to those seen in field incidents of bovine pneumonic pasteurellosis involving recently housed, weaned, single-suckled calves.

Pasteurella haemolytica A1 and bovine respiratory disease: pathogenesis.

The severe fibrinonecrotic pneumonia associated with pneumonic pasteurellosis usually results from colonization of the lower respiratory tract by Pasteurella haemolytica biotype A, serotype 1(A1). Despite recent research efforts, the authors lack a detailed understanding of the interactions and host response to P. haemolytica in the respiratory tract. The authors hypothesize that management and environmental stress factors or viral infection alters the upper respiratory tract (URT) epithelium allowing P. haemolytica to colonize the epithelium. Once the URT is colonized, large numbers of organisms enter the lung where they interact with alveolar macrophages. Endotoxin, released from the bacteria, crosses the alveolar wall where it activates pulmonary intravascular macrophages, endothelium, neutrophils, lymphocytes, platelets, complement, and Hageman factor leading to complex interactions of cells and mediators. It is the progression of this inflammatory response with neutrophil influx that is ultimately responsible for the pulmonary injury. Leukotoxin is a major virulence factor of P. haemolytica that allows it to survive by destroying phagocytic cells. At subcytolytic concentrations it may also enhance the inflammatory response by activating cells to produce mediators and release reactive oxygen metabolites and proteases.

Transit factors affecting shrink, shipping fever and subsequent performance of feeder calves.

Five shipments of feeder calves (965 head hauled in 11 drop-center trailers) were shipped 1,600 km by tractor trailer from Algood, Tennessee, to Bushland, Texas, during the fall seasons of 1976, 1977 and 1978. Shrink, incidence of shipping fever and subsequent feedlot performance of these feeder calves were analyzed. There were significant differences in shrink and subsequent feedlot performance between calves shipped on different dates. In only one instance was there a significant difference in shrink between trucks of steers shipped on the same date, and this was due to a difference of in-transit time between trucks. There were no significant differences in shrink, incidence of shipping fever of feedlot performance between calves shipped in different trailer compartments, nor were there any interactions between shipping dates and trailer compartments for shrink, incidence of shipping fever and feedlot performance. The number of calves treated for shipping fever did not differ significantly among trailer compartments, but did differ among shipment dates. Significant differences in morbidity between shipping dates indicate that the incidence of shipping fever is apparently affected by environmental conditions before, during and immediately after transit. The results indicate that multiple truckloads of calves, if traveling together, can be treated as a single unit for the statistical analysis of shrink, incidence of shipping fever and feedlot performance.

Induction of acute bronchopneumonia in mice by intrabronchial inoculation of Pasteurella haemolytica serotype 1.

Dose dependent pulmonary lesions of acute bronchopneumonia were induced in male, outbred Swiss Webster mice by intrabronchial inoculation of Pasteurella haemolytica. Five exponential dilutions ranging from 5 x 10(4) to 5 x 10(8) colony forming units per mL (CFU/mL) of Pasteurella haemolytica serotype 1 were inoculated into five groups of mice. Mice were killed by cervical dislocation 24 hours postinoculation. Pulmonary lesions occurred in mice of all five groups, however, 5 x 10(7) CFU/mL was the minimal dose which consistently produced lesions. Focal parenchymal necrosis, suppurative bronchiolitis, and flooding of interalveolar septa and alveoli by edema fluid, fibrin, neutrophils and macrophages, were observed microscopically. We conclude that outbred Swiss Webster mice can be used as a model for the study of selected disease mechanisms of acute lung inflammation and that this model may be used to determine some of the pathogenic mechanisms involved in the development of pulmonary lesions in bovine pneumonic pasteurellosis.

Treatment of experimentally induced pneumonic pasteurellosis of young calves with tilmicosin.

Twenty four (24) healthy male Holstein calves (< 70 kg) were each experimentally infected by intrabronchial inoculation of 4.0 x 10(9) viable cells of Pasteurella haemolytica-AI (B122) at Time = 0 h. At 1 h following inoculation animals received either: 1) Sham treatment with sterile 0.85% saline SC (n = 12); or 2) a single injection of 10 mg tilmicosin per kg body weight (n = 12). Calves that were non-infected and tilmicosin-treated were also included for determining tilmicosin concentrations in serum and lung tissue at 1, 2, 4, 6, 8, 24, 48, and 72 h (n = 3-per time). In the infected calves, response to therapy was monitored clinically. Serum samples were collected for determination of tilmicosin concentrations using HPLC. Any animal becoming seriously ill was humanely killed. Complete necropsy examinations were performed on all animals and included gross pathologic changes, bacteriologic analysis, histopathology, and determination of pulmonary concentrations of tilmicosin. Tilmicosin treated animals responded significantly better to therapy than saline-treated control calves. Clinical assessment of calves during the study indicated that tilmicosin-treated calves had significantly improved by T = 8 h compared to satine-treated animals (P < 0.05). At necropsy tilmicosin-treated calves had significantly less severe gross and histological lesions (P < 0.05) of the pulmonary tissue. Of the 12 saline-treated calves, 92% (11/12) had Pasteurella haemolytica-A1 in lung tissue, while of the tilmicosin-treated calves 0% (0/12) cultured positive for P. haemolytica. Mean (+/- standard error) serum tilmicosin concentrations in infected calves peaked at 1 h post-injection (1.10 +/- 0.06 micrograms/mL) and rapidly decreased to 0.20 +/- 0.03 microgram/mL, well below the MIC of 0.50 microgram/mL for P. haemolytica-A1 (B122), by 12 h. These serum concentrations were very similar to serum concentrations of tilmicosin in non-infected tilmicosin-treated calves. Lung tissue concentrations of the antibiotic were comparatively high, even at 72 h post-infection (6.50 +/- 0.75 ppm). Lung tissue concentrations at 72 h were significantly higher in experimentally infected calves than in non-infected tilmicosin-treated animals (P < 0.05). These data demonstrate that tilmicosin was effective in treating experimentally-induced pneumonic pasteurellosis as determined by alleviation of clinical signs, pathological findings at post mortem, and presence of viable bacteria from the lung. Concentrations substantially above MIC for P. haemolytica were present in lung tissue even at 72 h following a single subcutaneous injection of 10 mg tilmicosin per kg body weight.

Changes in blood and bronchoalveolar lavage fluid components in calves with experimentally induced pneumonic pasteurellosis.

Pneumonic pasteurellosis was experimentally induced in calves by inoculation of 5 x 10(8) Pasteurella haemolytica organisms into the right diaphragmatic lung lobe. Blood and bronchoalveolar lavage fluid samples were obtained prior to inoculation and at postinoculation hour (PIH) 2, 4, and 6. Calves developed acute lung injury, characteristic of pneumonic pasteurellosis. Lesions were found only in the right diaphragmatic lobe. By PIH 4, significant (P less than 0.01) increases were detected in lavage fluid total cell count, neutrophil count, total protein and albumin concentrations, and alkaline phosphatase (ALP) and lactic dehydrogenase (LD) activities. Myeloperoxidase and elastase activities did not increase. Neutrophil depletion ameliorated the lung lesions and prevented the increase in lavage fluid cell count, total protein, and albumin concentrations and ALP and LD activities. Treatment with the iron chelator, deferoxamine mesylatehydroxyethyl starch, attenuated the increase in total protein and albumin concentrations and ALP and LD activities at PID 4, but not PIH 6. Treatment with a neutrophil function inhibitor, pentoxifylline, prevented the increase in lavage fluid neutrophil numbers, but accentuated the increase in total protein and albumin concentrations, and ALP, LD, myeloperoxidase, and elastase activities.

Immunoperoxidase evaluation of the relationship between necrotic lesions and causative bacteria in lungs of calves with naturally acquired pneumonia.

An immunoperoxidase technique was used to study the relationship between the necrotic lesions and causative bacteria found in lungs of 53 calves that had naturally acquired pneumonia. Four types of necrotic lesions were identified on the basis of morphologic characteristics as follows: type 1 had coagulation necrosis surrounded by a dense zone of numerous degenerated leukocytes; type 2 was similar to type 1, but the central area of the lesions was severely affected, had no alveolar architecture remaining, and was surrounded by a thin, sparse layer of degenerated leukocytes; type 3 had small swirling accumulation of degenerated leukocytes; and type 4 had necropurulent lesions resembling abscesses. By use of the immunoperoxidase technique, Pasteurella haemolytica serovar 1 antigen was confirmed to be associated with the necrotic lesions in many cases of type 1 and in some cases of types 2 and 3. Although some lesions were induced by other bacteria (Haemophilus somnus or Actinomyces pyogenes), the pneumonic lesions associated with P haemolytica could be differentiated from other pneumonic lesions in calves by use of the immunoperoxidase technique.

Interaction of Pasteurella haemolytica with bovine neutrophils: identification and partial characterization of a cytotoxin.

Timed cultures of Pasteurella haemolytica 12296 strain in RPMI 1640 medium (with L-glutamine, pH 7.4) were used to determine the correlation between cytotoxin production and the age of the culture. Cytotoxic activity was measured by a 51Cr-release assay and trypan blue exclusion test with bovine neutrophils as target cells. Results demonstrated that optimal cytotoxin production occurred during the logarithmic phase (peaked at 6 hours) and decreased during the stationary phase of bacterial growth. The cytotoxin was concentrated by sequential ultrafiltration on Diaflo XM 50, XM 100, and XM 300 membranes. The cytotoxin was retained on an XM 300 membrane. These studies indicated that the molecular weight of cytotoxic substance was 300,000 or more. The cytotoxin was heat labile, oxygen stable, and susceptible to extremes of pH and killed bovine neutrophils and mononuclear leukocytes. It was not hemolytic to bovine or ovine RBC. The cytotoxic activity was inactivated by trypsin and did not contain any detectable endotoxin. Bovine fetal serum and serum collected before immunization from neonatal calves did not neutralize the cytotoxic effects of toxin on neutrophils. However, adult bovine serum from 6 cows and an antiserum (against the cytotoxin) neutralized the cytotoxin, as revealed by both the 51Cr-release assay and the trypan blue exclusion test. This was confirmed by transmission electron microscopy. These results indicated that the cytotoxin may be antigenic in cattle. The significance and implications of these findings to bovine pasteurellosis are discussed.

Pneumonic pasteurellosis induced experimentally in gnotobiotic and conventional calves inoculated with Pasteurella haemolytica.

Experimental pneumonia caused by Pasteurella haemolytica was induced in 2-week-old gnotobiotic (n = 4) and conventional (n = 6) calves by endobronchial inoculation into the right caudal lung lobe of 7.9 x 10(10) +/- 0.6 x 10(10) (mean +/- SD) colony-forming units of P haemolytica in the 6-hour log phase of growth. The calves were studied for 24 hours or less. Regression lines for the relationship between clinical index and time for the gnotobiotic group and conventional group of calves were compared, and the clinical index was found to be significantly (P less than or equal to 0.005) more rapid in the gnotobiotic group. There was also a significant difference in the preinoculation, absolute segmented neutrophil count (P less than or equal to 0.05), and in the total serum protein, albumin, and globulin values (P less than or equal to 0.05). Comparison of the preinoculation and post inoculation blood cell and blood chemical values revealed a significant increase (P less than or equal to 0.05) in the numbers of band neutrophils and fibrinogen in conventional calves, and a significant decrease (P less than or equal to 0.05) in the total WBC count in gnotobiotic calves. Necropsy of both groups of calves revealed a circular to oblong lesion that was congested, edematous, and firm, and which occupied 20% to 100% of the right caudal lung lobe and involved the remaining lung lobes to a more minor degree. When mean lesion scores of the 2 groups of calves were compared, no significant difference (P less than or equal to 0.05) was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical and pathologic studies of experimentally induced Pasteurella haemolytica pneumonia in calves.

Pasteurella haemolytica pneumonia of the right caudal lung lobe was experimentally induced in 2-week-old Holstein calves (n = 11) by endobronchial inoculation of 7.9 x 10(10) colony-forming units of 6-hour log-phase bacteria. Calves were studied for 72 hours after inoculation. The challenge procedure consistently induced a lesion in the right caudal lung lobe, which was consistent radiographically with results of pathologic examination and a similar volume of bronchography contrast medium. Clinically, the calves developed a significant increase in rectal temperature within 24 hours after inoculation. Seventy-two hours after inoculation, the total WBC counts, absolute band neutrophil counts, monocyte counts, and blood fibrinogen concentrations were significantly higher than normal and albumin concentration was significantly decreased. Necropsy revealed a circular to oblong lesion that was congested, edematous, and firm and occupied 20 to 40% of the right caudal lung lobe. Histologic examination revealed a severe acute inflammatory reaction characterized by cellular exudate and proteinaceous fluid in the alveoli, interlobular septa, and pleura.

Conglutinin and immunoconglutinin titers in stressed calves in a feedlot.

OBJECTIVE: To determine whether increased conglutinin titers are evident in stressed calves that do not develop respiratory tract disease in feedlots, compared with respiratory tract disease, and to determine the increase in immunoconglutinin titers. ANIMALS: 101 mixed-breed beef calves. PROCEDURE: Calves were processed at 4 farms of origin and allowed to remain with their dams for another 100 days. Calves from each farm were brought to a centrally located order-buyer barn. In a feedlot, 101 calves were assigned to pens and observed daily for clinical signs of acute respiratory tract disease. When sick calves were detected, they were treated with antibiotics and isolated in a pen for 4 days. Conglutinin and immunoconglutinin titers were determined for all calves. RESULTS: During the 28-day study, 73 calves developed respiratory tract disease, whereas 28 calves remained healthy. Mean conglutinin titers differed significantly among calves from the 4 farms. Significant differences were not detected in conglutinin titers among calves on the basis of sex, morbidity, or vaccination status against Mannheimia haemolytica at each farm, the order-buyer barn, or the feedlot on days 8, 15, and 28 after arrival. Immunoconglutinin titers in calves differed significantly among farms and morbidity status. CONCLUSIONS AND CLINICAL RELEVANCE: Mean conglutinin titers in calves do not appear to be associated with the incidence of acute respiratory tract disease; however, increased immunoconglutinin titers appear to be associated with recovery of stressed calves from respiratory tract disease during the first 15 days after arrival in a feedlot.

Respiratory tract immune response to microbial pathogens.

Effective resistance to respiratory tract infection depends principally on specific immunity on mucosal surfaces of the upper or lower respiratory tract. Respiratory tract immune response comprises antibody and cell-mediated systems and may be induced most readily by surface presentation of replicating agents but can result from parenteral or local presentation of highly immunogenic antigens. Upper and lower respiratory tract systems differ in immunologic competence, with the lungs having a greater inventory of protective mechanisms than the trachea or nose. Several effective vaccines have been developed for prevention or modification of respiratory tract diseases.

Therapy of bovine bacterial pneumonia.

Practical strategies for developing rational therapeutic regimens based on in vitro sensitivity and pharmacokinetic disposition are presented. Special attention is given to Pasteurella haemolytica, which is regarded as the most frequent cause of bovine bacterial pneumonia. Bacterial-dependent and host-dependent causes of therapeutic failure and potentially valuable novel therapies and drug combinations are considered.

Feedlot cattle pneumonia.

The etiology, epidemiology, clinical signs, and pathology of feedlot cattle pneumonias are discussed. This information enables a clinician with a feedlot cattle pneumonia problem to give prompt, useful advice on cause, prevention, and treatment based on findings of the feedlot visit.